Therapeutic Use of Botulinum Toxin
The clostridial neurotoxins consist of the botulinum toxins, the botulinum-like toxins and tetanus toxin. Botulinum neurotoxin is a protein molecule that is produced by the bacterium Clostridium botulinum, and it is considered to be the most deadly poison known. (Gill, M. D., "Bacterial toxins: a table of lethal amounts" Microbiol. Rev. (1982) 46: 86-94.) Clostridium botulinum is the species name assigned to four metabolically diverse groups of anaerobic bacteria whose one common feature is the production of botulinum neurotoxin. Seven different antigenic variants of the botulinum neurotoxin molecule are presently known and are serologically distinguishable from each other by means of monovalent antitoxin antibodies. These different toxin types have arbitrarily been assigned the letters A through G. Botulinum toxin produces muscle paralysis and relaxation by blocking the motoneuron from releasing acetylcholine at the neuromuscular junction. This effect derives from the enzymatic action of the "light" (50,000 MW) chain of botulinum toxin, the various types (A-G) of which hydrolyze key proteins which the motoneuron relies on for the release of the acetylcholine-containing vesicles that trigger muscle contraction. In addition, the "light" chain of tetanus neurotoxin is a protease that acts identically to botulinum type B. (Huttner, W. B., "Snappy Exocytoxins" Nature (1993) 365: 104-105). The substrate proteins for the clostridial neurotoxins are components of the synaptic vesicle "docking" or "fusion" complex and are known by their acronyms VAMP, SNAP-25 and syntaxin (Barinaga, M., "Secrets of Secretion Revealed" Science (1993) 260: 487-489). The relationships between these substrates and the botulinum and tetanus toxins are shown in Table 1 below.
TABLE 1 ______________________________________ Light Chain Substrates (1) (2) (3) VAMP SNAP-25 Syntaxin ______________________________________ type B type A type C type D type E type F type G tetanus ______________________________________
The properties of botulinal toxins have allowed them to be used therapeutically. Botulinum toxin is used to produce a temporary muscle paralysis in diseases characterized by: 1) overactivity of a particular muscle or muscle group (e.g., strabismus); 2) involuntary muscle spasm (the dystonias); and 3) other disorders of movement. Numerous therapeutic uses for botulinum toxin were addressed at a November 1990 National Institutes of Health Consensus Development conference. The consensus panel from this conference resolved that botulinum toxin therapy is safe and effective for treating strabismus, blepharospasm, hemifacial spasm, adductor spasmodic dysphonia, jaw-closing oromandibular dystonia, and cervical dystonia. (Clinical Use of Botulinum Toxin. (Reprinted from NIH Conses. Dev. Conf. Consens. Statement 1990 Nov 12-14; 8(8)) Because the effects of the toxin last for only a few months, repeated injections of toxin are necessary to sustain its therapeutic benefit for chronic conditions.
In December 1989, the U.S. Food and Drug Administration (FDA) licensed for medicinal use a crystalline preparation of botulinum type A toxin, OCULINUM.RTM. toxin (also designated BOTOX.RTM. toxin, Allergan, Inc., Irvine, Calif.). OCULINUM.RTM. toxin contains botulinum neurotoxin, other bacterial protein molecules that co-crystallized with the neurotoxin, and stabilizing materials. OCULINUM.RTM. toxin is typically used to treat diseases such as strabismus, blepharospasm, hemifacial spasm, adductor spasmodic dysphonia, jaw-closing oromandibular dystonia and cervical dystonia.
Because the various botulinum and tetanus toxin light chains (proteases) have different substrates within the motoneuron cytosol, several two-fold and three-fold type combinations are therapeutically beneficial. There are three groupings of two-fold combinations. First, any toxin listed in column (1) of the above table can be combined with any toxin listed in column (2); second, column (3) (i.e., type C toxin) can be combined with any toxin in column (1); and third, column (3) can be combined with any toxin in column (2). The beneficial three-fold combinations all contain type C toxin. Type C toxin can be combined with either type A or type E toxin and the resulting combination can then be combined with any of the toxins in column (1). These 10 combinations consist of ABC, ADC, AFC, AGC, A-tetanus-C, EBC, EDC, EFC, EGC, and C-E-tetanus.
Additionally, a number of organisms producing botulinum-like toxins have been identified. For example, a unique strain of Clostridium baratii produces a type F-like toxin, and a unique strain of Clostridium butyricum produces a type E-like toxin.
Initially it was believed that individuals exposed to botulinum toxin did not produce antibodies against the toxin, due to the phenomenal potency of the toxin. It was thought that an immunogenic dose of the toxin would be lethal, i.e., that the amount of toxin needed to induce antibody production exceeded the lethal dose. This belief derived from decades of experience with foodborne botulism.
Clostridium botulinum and its toxin were first described as the cause of foodborne botulism in 1897. (Van Ermengem E., "Ueber einem neuen anaeroben Bacillus und seine Beziehungen zum Botulismus" Z. Hyg. Infektionskrankh. (1897): 26: 1-26. English translation, Rev. Infect. Dis. (1979) 1: 701-19.) Based on the experience with foodborne botulism, it had been determined that no antibodies developed in patients who survived the illness, even among patients who were so ill that they required mechanical ventilation for survival. (Koenig, M. G., et al., "Clinical and Laboratory Observations on Type E Botulism in Man" Medicine (1964) 43: 517-45) Consistent with this understanding, it had been reported that patients who recovered from either type B or type E foodborne botulism later experienced a second occurrence of foodborne botulism caused by the same toxin type. (Beller, M. and Middaugh, J. P., "Repeated type E botulism in an Alaskan Eskimo" N. Engl. J. Med. (1990) 322: 855; Schroeder, K., Tollefsrud, A. L., "Botulism from Fermented Trout" T. Norske Laegeforen (1962) 82: 1084-87) These reports were used to support the conclusion that exposure to minute, disease-causing amounts of botulinum toxin did not result in the development of antibodies to the toxin.
The failure of the immune system to make antibodies when exposed to botulinum toxin through illness was considered to be analogous to the experience with the human illness tetanus. Tetanus results from the effects of a neurotoxin (tetanospasmin) produced in infected wounds by Clostridium tetani, a member of the same bacterial genus as Clostridium botulinum. Of all known toxins, tetanospasmin is second only to botulinum toxin in potency. (Gill, M. D., "Bacterial Toxins: A Table of Lethal Amounts" Microbiol. Rev. (1982) 46: 86-94) Experience with tetanus had shown that "the quantity of tetanospasmin required to produce tetanus is insufficient to induce a protective immune response, and patients with this disease require a primary immunization series." (Mandell, G. L., Douglas, R. G., Bennett, J. E. eds., Principles and Practice of Infectious Diseases, 3d ed., Churchill Livingstone, N.Y., (1990) at p. 1845). Thus, with botulism as with tetanus, it was understood that an immunogenic dose of toxin exceeded the lethal dose.
However, in the context where botulinum toxin is used therapeutically, a new picture has developed. It has been observed that some patients who initially benefitted from the toxin, later became insensitive (refractory, resistant) to its use. This insensitivity has been attributed to the development, upon repeated injections with the toxin, of antibodies against the toxin.
Evidence that patients were developing neutralizing antibodies against the toxin after repeated treatments, thereby becoming unresponsive to the therapeutic effects of the toxin, began to emerge in the late 1980's. Brin and colleagues in 1988 reported that two of 90 patients they studied had developed antibodies to botulinum toxin and had become refractory to treatment (Brin, M. F., et al., "Localized Injections of Botulinum Toxin for the Treatment of Focal Dystonia and Hemifacial Spasm" Adv. Neurol. (1988) 50: 599-608) Jankovic and Schwartz obtained sera from 14 patients characterized as "non-responders" to botulinum toxin therapy, and found neutralizing antibodies against the toxin in 5 (37.5%); no antibodies were found in 32 patients characterized as "responders" to the toxin (P&lt;0.0001). (Jankovic, J., Schwartz, K. S., "Clinical correlates of response to botulinum toxin injections" Arch. Neurol. (1991) 48: 1253-56) The patients with antibodies had, on average, received approximately twice as much toxin [1600 U, range 500-2450] as had the patients without antibodies [891 U, range 100-2150]. Additionally, Scott identified seven dystonia patients who had become refractory to treatment; all had neutralizing antibodies present in their sera. (Scott, A. B., "Clostridial Toxins as Therapeutic Agents" pp. 399-412 in Simpson, L. L., ed. Botulinum neurotoxin and tetanus toxin, Academic Press, NY (1989)) Six of the patients in the Scott study had received 300-400 ng and one only 100 ng of toxin within a 30-day period. (For clinical purposes, 1 ng of OCULINUM.RTM. toxin equals approximately 2.5-3.0 U.)
In England, Hambleton and colleagues studied 20 patients categorized as "maintained response" or as "diminished response." (Hambleton, P., Cohen, H. E., Palmer, B. J., Melling, J., "Antitoxins and botulinum toxin treatment" Brit. Med. J. (1992) 304: 959-60 at 959) These patients were selected from a group of several hundred spasmodic torticollis patients who had been treated for several years with botulinum type A toxin. Seven (35%) of the patients studied were found to have toxin-neutralizing antibodies that considerably diminished or abolished their therapeutic response to the toxin.
The American and British findings are especially notable when taken together, since the British investigators used a preparation of botulinum toxin that was made in England, in contrast to the preparation that is both made and used in the United States. Hence, neutralizing antibodies have arisen in patients irrespective of whether the British or American preparation of botulinum toxin was used.
Heretofore, the research emphasis concerning the therapeutic use of botulinum toxin has focused on development of more highly purified toxins as a means to control the immune response.
The focus on developing more highly purified toxins has been noted by two editorials from late 1992, editorials that overviewed the therapeutic use of botulinum toxin. One editorial appeared in the Dec. 19/26, 1992 issue of the Lancet ("Botulinum Toxin" Lancet (1992) 2: 1508-9), and the other was published Nov. 14, 1992 in the British Medical Journal (Lees, A. J., "Botulinum Toxin: Useful in Adult Onset Focal Dystonias" BMJ (1992) 305: 1169-70 at p.1170).
The Lees editorial, noted that "[p]atients have continued to respond with benefit for more than five years, although antibodies to the toxin may develop in the peripheral blood, leading to initial unresponsiveness or late resistance (Lees, A. J., "Botulinum Toxin: Useful in Adult Onset Focal Dystonias" BMJ (1992) 305: 1169-70 at p. 1170). The Lees editorial concluded stating, "Trials of other types of botulinum toxin are under way, and more effective toxins capable of producing longer durations of benefit without inevitably increasing unwanted effects may be developed in the near future." Thus, the proposed solution to the problem of antibody formation and resultant insensitivity was to increase the purity of the toxins used or to develop the other botulinum toxin serotypes (e.g., B, C, D etc.).
The Lancet editorial states "Although the toxin moiety itself is known to be antigenic, toxin neutralizing antibodies could also arise from other parts of the BtA-hemagglutinin complex, so a different preparation of BtA [botulinum toxin type A] might be worth trying. A purer form of BtA would allow us to explore this possibility." (Editorial, "Botulism Toxin" Lancet (1992) 3: 1508-09 at p.1508). Again, the proposed solution to the problem of antibody formation was to increase the purity of the toxin preparations injected into patients.
Notably, neither of these reviews discloses nor suggests the possibility of using 1) a human-derived botulism immune globulin; 2) which immune globulin would be injected intravenously; 3) in order to prevent the unwanted side effects of toxin diffusion and antitoxic antibody formation in treated patients.
A detailed discussion regarding the development of antibodies to botulinum toxin in toxin-treated patients was reported by Hatheway and Dang. (Hatheway, C. L., Dang, C., "Immunogenicity of the Neurotoxins of Clostridium botulinum" in Jankovic, J., Hallet, M. eds. Therapy with Botulinum Toxin, Marcel Dekker, New York, N.Y. (1993)) Eighty-eight patients in the U.S. were followed for one year after they began treatment with toxin, during which time the amount of toxin received ranged from 0 to 2550 units. At one year into treatment, 29 patients (33%) had developed neutralizing antibody against the toxin. The antibody-positive patients had received an average of 1051 t.u. of toxin, while the antibody-negative patients had received an average of 301 t.u., again suggesting a dose-response effect in the induction of antibody. This dose-response possibility was borne out when the patients were stratified according to dose received: &lt;500 treatment units, 4% with antibody; 500-1000 treatment units, 45% with antibody; 1000-2000 treatment units, 83% with antibody; &gt;2000 treatment units, 100% with antibody. In addition, Hatheway and Dang noted that continued treatment of patients who have subdetectable levels of antibodies might serve to boost the antitoxin titers above the minimum demonstrable level.
Patients who require botulinum toxin injections generally must have the injections repeated at regular intervals. Dose-response data, such as that of Hatheway and Dang, suggest that as the duration of currently practiced botulinum toxin treatment is extended, more patients will develop antibody and thereby lose the therapeutic benefit of the toxin. For this reason some expert physicians recommend limiting patients with dystonia to four injections per year, even if the beneficial effect of injected botulinum toxin lasts less than three months (Lees, A. J. et al., "Treatment of Cervical Dystonia Hand Spasms and Laryngeal Dystonia with Botulinum Toxin" J. Neurol. (1992) 239: 1-4).
Antibody development also limits the potential to administer combinations of botulinum toxin serotypes. In this regard, the current practice holds that patients should be treated with just one toxin type at a time, so that if and when antibodies to that toxin type develop, the patient can be changed to a different, single toxin type. To illustrate, patients who had been treated with botulinum type A toxin and developed neutralizing antibodies to it were shifted to treatment with botulinum type F toxin (Greene, P. E. and Fahn, S., "Use of Botulinum Toxin Type F Injections to Treat Torticollis in Patients with Immunity to Botulinum Toxin Type A" Movement Disorders (1993( 8: 479-83).
Although the problem of antibody development with botulinum toxin therapy has not been successfully addressed, other problems with the toxin therapy have been studied. It has been noted that patients injected with botulinum toxin have suffered complications due to the apparent diffusion of the toxin from the injected muscle(s) to adjacent muscles. For example, complications have included drooping eyelids (to the extent that vision is blocked), and difficulty with swallowing (to the extent that hospitalization was needed in order that a stomach feeding tube could be placed). (Jankovic, J., Brin, M. F., "Therapeutic Uses of Botulinum Toxin" N. Engl. J. Med. (1991) 324: 1186-94; Schantz, E. J., Johnson, E. A., "Properties and Use of Botulinum Toxin and other Microbial Neurotoxins in Medicine" Microbiol Revs. (1992) 56: 80-99) In certain clinical situations, such as with small or vitally-placed muscles, diffusion (or "leaking" of toxin) has limited the amount of toxin that could otherwise have been therapeutically injected, because of concern that such side-effects would develop. (Clinical Use of Botulinum Toxin. (Reprinted from NIH Conses. Dev. Conf. Consens. Statement 1990 Nov. 12-14; 8(8) ); Jankovic, J., Brin, M. F., "Therapeutic Uses of Botulinum Toxin" N. Engl. J. Med. (1991) 324: 1186-94; Scott, A. B., "Clostridial Toxins as Therapeutic Agents" pp. 399-412 in Simpson, L. L., ed. Botulinum Neurotoxin and Tetanus Toxin, Academic Press, NY (1989); Scott, A. B., "Antitoxin reduces botulinum side effects" Eye (1988) 2: 29-32)
In certain situations the amount of botulinum toxin that can be injected is limited by anatomical considerations and the tendency of the injected toxin to diffuse away from the injection site. In particular, injection of toxin into dystonic muscles in the upper neck or in the back of the tongue must be limited in order to avoid paralyzing the gag reflex and the swallowing muscles. If these muscles are made flaccid by toxin that has diffused into them, then the patient may become unable to eat or unable to keep oral secretions from draining into the lungs. Also, the amount of toxin that can be injected into facial or eye muscles (e.g., for blepharospasm) is limited by the toxin's ability to diffuse into adjacent muscles (e.g., eyelid or oculomotor). When such diffusion occurs, the resulting muscle paralysis can cause double vision or ptosis so severe that sight is obstructed by the drooping eyelid. In general, these complications are considered unacceptable.
An experimental attempt to overcome the side-effect of toxin diffusion into adjacent muscles was attempted by Scott. (Scott, A. B., "Antitoxin reduces botulinum side effects" Eye (1988) 2: 29-32) Scott's effort utilized direct intramuscular injection of equine botulinum antitoxin. The antitoxin was injected into the toxin-treated muscles or into untreated adjacent eye muscles. At the time Scott did his clinical studies with antitoxin, the botulinum antitoxin was a horse-derived product available from Connaught Laboratories (Toronto, Canada).
In Scott's method of direct intramuscular injection of horse-derived antitoxin, Scott addressed only one of the fundamental problematic issues with botulinum toxin therapy: diffusion of toxin to adjacent muscles. Antibody development consequent to botulinum toxin use was neither contemplated nor addressed. Because of clinically observed weakness in muscles adjacent to those injected, Scott mentioned a theoretical possibility of intramuscular use of human-derived antitoxin: "A human-derived ATX [antitoxin] and the non-toxic large fragment of the toxin molecule to block unwanted toxin binding are additional related techniques to reduce side effects and to increase efficacy which we are pursuing and which avoid the theoretical risks of immunity or sensitisation [sic] to equine-derived proteins" (Scott A. B., "Antitoxin Reduces Botulinum Side Effects" Eye (1988) 2: 29-32 at p. 32) Again, however, this proposal for further study was set out in the context of intramuscular antitoxin injection, with a goal of controlling toxin diffusion.
Although Scott might have been interested in using human botulism immune globulin (BIG) for intramuscular injections, he was unable to do so. At the time of Scott's study, the U.S. Army had the world's only supply of BIG. Also, at that time, it was not yet known that some patients injected therapeutically with botulinum toxin would develop antibodies to it. Scott's concern addressed only the possibility of development of antibodies to the equine botulism antitoxin.
Scott's approach was clinically unsatisfactory because it required the injection of additional muscles besides those targeted for the toxin (requiring additional physician/patient time and risk), and because the horse-derived botulinum antitoxin was a foreign protein capable of stimulating antibody production against itself when injected into humans. Hence, with repeated use, patients given the horse-derived antitoxin can be expected to develop antibodies against it and also to become refractory to its effects, just as some patients have become refractory to the effects of injected botulinum toxin. Of further concern, the horse-derived botulinum antitoxin is known to provoke severe allergic complications when used to treat patients with food-borne botulism: approximately one in eight such patients experienced anaphylaxis or serum sickness (i.e., allergic shock or kidney damage). (Black, R. E., Gunn, R. A., "Hypersensitivity Reactions Associated with Botulinal Antitoxin" Am. J. Med. (1980) 69: 567-70)
In addition to its accepted usage for the treatment of strabismus and various dystonias, botulinum toxin has also been used to reduce facial wrinkles by temporarily weakening the underlying muscles. (Carruthers, J. D. A. and Carruthers, J. A., "Treatment of Glabellar Frown Lines with C. botulinum-A exotoxin" J. Dermatol. Surg. Oncol. (1992) 18: 17-21) If this cosmetic procedure finds widespread use, then based on the incidence of the dystonias in the general population, it is predicted that among the population cosmetically treated with botulinum toxin, some individuals will eventually experience the onset of a dystonia. For these patients to then be able to therapeutically benefit from injection of botulinum toxin, it is important that they would not have developed neutralizing antibodies against the toxin during their cosmetic treatment with it.
Therapeutic Use of Chimeric Toxins, Recombinant Toxins and Immunotoxins
Chimeric toxins, recombinant toxins, and immunotoxins are a relatively new group of macromolecules that are being developed for use in a variety of human illnesses. The underlying therapeutic principle is the joining of a toxin molecule to a targeting molecule of high specificity. The targeting molecule then delivers the toxin to the unwanted cell or tissue, where the toxin portion of the molecule is internalized and poisons the cell. Chimeric toxins, recombinant toxins, and immunotoxins are potentially useful in the treatment of cancer (both solid tumors and hematological malignancies), autoimmune diseases (e.g., rheumatoid arthritis and diabetes mellitus type 1), and other conditions such as Acquired Immunodeficiency Syndrome (AIDS), graft versus host disease (GVHD), vascular restenosis, and rejection of organ transplants (Vitetta, E. S. et al., "Immunotoxins: Magic Bullets or Misguided Missiles? Immunology Today (1993) 14: 252-259; Biro, S. et al., "In vitro Effects of a Recombinant Toxin Targeted to the Fibroblast Growth Factor Receptor on Rat Vascular Smooth Muscle and Endothelial Cells" Circ. Res. (1992) 71: 640-5; and Wawrzynczak, E. J. and Derbyshire, E. J. "Immunotoxins: the Power and the Glory" Immunology Today (1992) 13: 381-383).
Targeting molecules utilized to convey various toxins include monoclonal antibodies and antibody fragments, growth factors (e.g., epidermal growth factor), cytokines (e.g., interleukin-2), and plant lectins. The principal toxins (or their fragments) used currently are bacterial or plant in origin and include ricin, diphtheria toxin and Pseudomonas exotoxin A (Vitetta, E. S. et al., "Immunotoxins: Magic Bullets or Misguided Missiles? Immunology Today (1993) 14: 252-259; and Wawrzynczak, E. J. and Derbyshire, E. J. "Immunotoxins: the Power and the Glory" Immunology Today (1992) 13: 381-383). Other plant and fungal toxins (e.g., gelonin, saporin) known as "ribosome-inactivating-proteins" have had their genes cloned in preparation for possible use as immunotoxins. Ribosome-inactivating proteins are advantageous toxins because they are single-chain, low molecular weight proteins (Wawrzynczak, E. J., "Systemic Immunotoxin Therapy of Cancer: Advances and Prospects" Br. J. Cancer (1991) 64: 624-630). A chimeric toxin comprised of two distinct toxins from different bacterial species has been evaluated experimentally (Prior, T. I. et al., "Barnase Toxin: A New Chimeric Toxin Composed of Pseudomonas Exotoxin A and Barnase" Cell (1991) 65: 1017-23). One interesting immunotoxin used clinically contained a radioisotope as its passenger toxin (Zeng, Z C et al., "Radioimmunotherapy for Unresectable Hepatocellular Carcinoma Using I.sup.131 -Hepama-1 mAb: Preliminary Results" J. Cancer Res. Clin. Oncol. (1993) 119: 257-9).
Although immunotoxins are still in the early stages of clinical evaluation as therapeutic agents, a number of problems that limit their utility have become evident. These problems include the complications and side-effects of 1) immunogenicity, 2) hepatotoxicity, 3) cross-reactivity with and injury to normal tissue (e.g., stomach, nerve, muscle), 4) injury to vascular endothelium, resulting in the "vascular leak" syndrome, and 5) instability, resulting in free toxin in the body. In addition, treatment of solid tumors (e.g., lung, breast, liver) with immunotoxins has been less satisfactory than treatment of hematological malignancy (e.g., leukemia) because of practical problems related to the large bulk of solid tumors and the anatomical difficulty of delivering an immunotoxin molecule to all tumor cells. New methods that overcome or circumvent these problems are needed if immunotoxins are to become useful therapeutic tools.
A variety of approaches to overcome these clinical obstacles have been tried. The instability that results from using a chemical linkage (e.g., sulfhydryl bonding) between toxin and antibody has been improved by moving to the covalent peptide-bond linkage of the recombinant toxin. Potential cross-reactivity is minimized by selecting the most highly specific antibody or lectin available, after screening in vitro in rodents and in primates. Nonetheless, cross-reactivity of immunotoxins with normal tissues remains a clinical problem. In the case of ricin A-chain toxins, hepatotoxicity resulted from toxin-associated oligosaccharides binding directly to liver cells and was circumvented by eliminating the oligosaccharides from ricin. Bacterial toxins and ribosome-inactivating-proteins cause hepatotoxicity by binding to non-carbohydrate hepatocyte receptors or by binding to serum proteins that have receptors in the liver, and solutions to this form of hepatotoxicity have not yet been found. To improve the efficacy of immunotoxins against solid tumors, targeting of the immunotoxin antibody to unique antigens expressed in the tumor's vasculature, with consequent interruption of the tumor's blood supply, has been accomplished inexperimental animals (Burrows, F. J. and Thorpe, P. E., "Eradication of Large Solid Tumors in Mice with an Immunotoxin Directed Against Tumor Vasculature" Proc. Natl. Acad. Sci. USA (1993) 90: 8996-9000).
The "vascular leak syndrome" consists of edema, decreased serum albumin and weight gain that results either directly or indirectly from immunotoxin-mediated injury to the vascular endothelium and increased vascular permeability. When the leakage occurs in the lungs, the resultant pulmonary edema can be life-threatening (Amlot, P. L. et al., "A Phase I Study of an Anti-CD22-Deglycosylated Ricin A Chain Immunotoxin in the Treatment of B-cell Lymphomas Resistant to Conventional Therapy" Blood (1993) 82: 2624-33). In an experimental system using human umbilical endothelial cells, "rapid and dramatic" morphological changes consisting of cell rounding with gap formation were seen one hour after exposure to a ricin-A chain immunotoxin, while inhibition of protein synthesis was not observed until four hours after exposure (Soler-Rodriguez, A. M. et al., "Ricin A-Chain and Ricin A-Chain Immunotoxins Rapidly Damage Human Endothelial Cells: Implications for Vascular Leak Syndrome" Exp. Cell Research (1993) 206: 227-34). There is presently no circumvention for the "vascular leak syndrome," in part because its mechanism remains obscure.
A major limitation to the effectiveness of immunotoxin therapy is the problem of immunogenicity. Patients treated with immunotoxins rapidly develop their own antibodies against one or both (usually both) portions of the immunotoxin molecule, often within two weeks of starting immunotoxin therapy (Pai, L. H. and Pastan, I., "Immunotoxin Therapy for Cancer" JAMA (1993) 269: 78-81; and Skolnick, A. A., "First Immunotoxin Therapy for Many Common Solid Tumors Enters Phase I Clinical Trial" JAMA (1993) 270: 2280). A recent summary of 15 clinical trials with immunotoxins in which antibody induction was studied determined that in 12 (80%) of the trials, at least 50% of patients developed antibodies against the immunotoxin being evaluated. In four of the clinical trials more than 90% of the patients developed antibodies (Vitetta, E. S. et al., "Immunotoxins: Magic Bullets or Misguided Missiles? Immunology Today (1993) 14: 252-259).
Immunotoxin treatment needs to be given repeatedly in order to maximize tumor regression and eradication (Friedman, P. N. et al., "Antitumor Activity of the Single-Chain Immunotoxin BR96 sFv-PE40 Against Established Breast and Lung Xenografts" J. Immunol. (1993) 150: 3054-61; Skolnick, A. A., "First Immunotoxin Therapy for Many Common Solid Tumors Enters Phase I Clinical Trial" JAMA (1993) 270: 2280; and Wawrzynczak, E. J., "Systemic Immunotoxin Therapy of Cancer: Advances and Prospects" Br. J. Cancer (1991) 64: 624-630). When endogenous antibody formation occurs, the efficacy of immunotoxin treatment is substantially diminished or negated. This decreased efficacy is thought to result from the increased rate of clearance of the immunotoxin or from blocking of the receptor site or toxic activity site of the immunotoxin (Vitetta, E. S. et al., "Immunotoxins: Magic Bullets or Misguided Missiles? Immunology Today (1993) 14: 252-259; and Wawrzynczak, E. J. and Derbyshire, E. J., "Immunotoxins: the Power and the Glory" Immunology Today (1992) 13: 381-383).
Various approaches to overcome the problem of immunogenicity have been tried but without success. Immunosuppressive drugs such as cyclophosphamide, prednisone, azathioprine and cyclosporin A failed to prevent patients from developing endogenous antibody in the face of repeated administration of immunotoxin (Wawrzynczak, E. J., "Systemic Immunotoxin Therapy of Cancer: Advances and Prospects" Br. J. Cancer (1991) 64: 624-630). In experimental animals modification of a Pseudomonas exotoxin-derived immunotoxin with monomethoxy-polyethylene glycol (mPEG) diminished immunogenicity 5- to 10-fold, prolonged circulation time and preserved its anti-tumor effect (Wang, Q. C. et al., "Polyethylene Glycol-Modified Chimeric Toxin Composed of Transforming Growth Factor Alpha and Pseudomonas Exotoxin" Cancer Research (1993) 53: 4588-94). "Humanizing" the mouse-cell-derived monoclonal carrier antibody has been seen as a possible solution to the problem of immunogenicity (Skolnick, A. A., "First Immunotoxin Therapy for Many Common Solid Tumors Enters Phase I Clinical Trial" JAMA (1993) 270: 2280; and Winter, G and Harris, W. J., "Humanized Antibodies" Immunol. Today (1993) 14: 243-246). However, the recently accomplished replacement in mice of the mouse genes for antibody production with the human genes for antibody production may portend an unlimited supply and variety of fully human-compatible antibodies from mice (Green, L. L. et al., "Antigen-Specific Human Monoclonal Antibodies from Mice Engineered with Human Ig Heavy and Light Chain YACs" Nature Genetics (1994) 7: 13-21; and Lonberg, N. et al., "Antigen-Specific Human Antibodies from Mice Comprising Four Distinct Genetic Modifications" Nature (1994) 368: 856-859). Another effort to make immunotoxins less immunogenic used mouse/human antibody and a human-homolog RNase gene to create a novel "humanized" immunotoxin (Rybak, S. M. et al., "Humanization of Immunotoxins" Proc. Natl. Acad. Sci. (1992) 89: 3165-9). Because the current art has not solved the immunogenicity problem, a simple, safe and comfortable method to minimize or abolish the immunogenicity of immunotoxins in patients undergoing treatment with them is still needed.
Passive Immunization
Antibodies have been given to patients in order to achieve passive immunization. The antibodies may be obtained from human or animal donors who have recovered from an infectious disease or have been immunized. This antibody product can be either whole serum or fractionated concentrated immune (gamma) globulin, which is predominantly IgG. These antibodies can provide immediate protection to an individual deficient in such antibodies.
When antibodies are obtained from animals, the animal sera give rise to an immune response that leads to rapid clearance of the protective molecules from the circulation of the human recipient. Additionally, animal sera provide a risk of allergic reactions, particularly serum sickness or anaphylaxis.
With regard to human antibodies, special preparations of human immune globulin with a high titer of a specific antibody are available. These preparations are obtained by hyperimmunizing adult donors or by selecting plasma which was tested for a high specific antibody content. Although the side effects of human immune globulin are minimal, its intramuscular administration is painful and, although rare, anaphylactoid reactions have been described.
Passive immunization has been carried out for infectious and noninfectious diseases. As an example of a noninfectious disease treated with passive immunization, Rh-negative persons are at risk of developing anti-Rh antibodies when Rh-positive erythrocytes enter their circulation. For Rh-negative women, this occurs regularly during a pregnancy with an Rh-positive fetus. Development of anti-Rh antibodies by a mother threatens all subsequent Rh-positive fetuses with erythroblastosis fetalis and death. This scenario can be prevented by administration of Rh immune globulin (RhIG) to the Rh-negative mother. RhIG is produced by having Rh-negative volunteers (originally men or nuns, because these women did not plan to have children) be injected with Rh-positive red cells to induce antibodies. Then, these volunteers are plasmapheresed to harvest the immune plasma, which is then processed into RhIG.
By use of RhIG, erythroblastosis fetalis is avoided in future Rh-positive fetuses. Passive immunization with Rh immune globulin (RhIG) suppresses the mother's normal immune response to any Rh-positive fetal cells that may enter her circulation. Passive immunization with RhIG may also protect in a nonspecific manner, analogous to the `blocking` effect of high-dose IgG in ameliorating autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP). With ITP, the beneficial blocking effect is thought to derive from the ability of the infused antibody to bind to receptors in the spleen and to prevent that organ from destroying the platelets to which the host's own "autoimmune" antibodies have become adherent (Berkman, S. A., et al., . "Clinical Uses of Intravenous Immunoglobulins" Ann. Int. Med. (1990) 112: 278-292).
Passive immunization can be carried out in another context that relates to Rh isoimmunization. Rh isoimmunization may occur consequent to blood transfusion. Most transfusion reactions to Rh can be prevented by transfusing Rh-negative individuals with Rh-negative blood. Of the Rh antigens, the D antigen is a high-incidence, strongly immunogenic antigen, approximately 50 times more immunogenic than the other Rh antigens. Thus, when determining Rh status, transfusion blood is typed routinely for D, but+not for other Rh antigens. However, immunization to other Rh antigens may occur even when Rh-negative blood is given to Rh-negative patients, since donor blood is not routinely typed for the non-D Rh antigens. Additionally, immunization and antibody formation to Rh antigens can occur in Rh-negative individuals due to transfusion errors. RhIG can be used to passively immunize and protect individuals from such situations. RhIG addresses a spectrum of Rh antigens because of the way it is made, utilizing red cells that contain an array of Rh antigens. Thus, the resulting RhIG is directed to various Rh antigens, in addition to the Rh D antigen.
Accordingly, Rh immunization can now be suppressed almost entirely if high-titer anti-Rh immunoglobulin (RhIG), available under the tradename RHOGRAM.RTM. for (Ortho Pharmaceuticals, Raritan, N.J.), is administered within 72 hours of the time the potentially sensitizing dose of Rh-positive cells were given.
As is the case with RhIG administration to pregnant Rh-negative women, the protective mechanism by which RhIG administration prevents development of Rh antibodies in Rh-negative individuals is not clear. RhIG does not effectively block Rh antigen from immunoresponsive cells by competitive inhibition, since it is known that effective doses of RhIG do not cover all Rh antigen sites on the fetal (or wrongly transfused) erythrocytes. Intravascular hemolysis with rapid clearance of erythrocyte debris by the reticuloendothelial system is also unlikely. Rather, after the Rh-positive cells are removed from the circulation, the RhIG-induced erythrocyte hemolysis is believed to be extravascular, primarily by phagocytic cells in the spleen and, to a lesser extent, the lymph nodes. The most likely therapeutic mechanism resulting from RhIG administration is a negative modulation of the primary immune response. It is believed that antigen-antibody complexes become bound to lymph node and splenic cells that have Fc receptors. These lymph node and spleen cells presumably then stimulate suppressor T cell responses, which subsequently prevent antigen-induced B cell proliferation and antibody formation.